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JEuropaisches Patentamt
European Patent Office © Publication number: 0 322 094

Office europeen des brevets A1

C12N 1/18 , C12P 21/02 ,
@ Date of filing: 25.10.88 //C12N5/00

The microorganism(s) has (have) been deposited

with i no iNSuonai L/Oiiecuon ot inousuiai ana

pacHo fViurt Castle Boulevard

Marine Bacteria under number NCIB 12242

Nottingham NG7 1FD(GB)

The title of the invention has been amended

(Guidelines for Examination in the EPO, A-lll,

11 South Road

7.3).

West Brldgford Nottingham NG2 7AG(GB)

inventor: Hinchliffe, Edward

16 Lambley Lane

Burton Joyce Nottingham NG14 5BG(GB)

Inventor Gelsow, Michael John

115 Main Street

28.06.89 Bulletin 89/26

East Brldgford Nottingham NG13 8NH(GB)

Inventor: Senior, Peter James

Wilson House Wilson

AT BE CH DE ES FR GB GR IT LI LU NL SE

Near Melbourne Derbyshlre(GB)

ERIC POTTER & CLARKSON 14 Oxford Street

Nottingham NG1 5BP(GB)

© Polypeptides corresponding to mature human serum albumin residues 1 to n, where n is between 369 and
419 inclusive, are useful as substitutes for albumin in the treatment of burns and shock in humans, the
clearances of undesirable compounds, (such as bilirubin) from human blood, in laboratory growth media and in
HSA assays.

HSA (1-389) is particularly preferred, although not novel per se.

The polypeptides may be produced by recombinant DNA techniques, especially in yeast

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EP 0 322 094 A1

POLYPEPTIDES

This invention relates to a novel polypeptide molecule which can be produced by recombinant DNA
technology and can be used for many of the existing applications of human serum albumin.

Human serum albumin (HSA) is the most abundant plasma protein, contributing 60% w/w of the total
protein content of the plasma. A molecule of HSA consists of a single non-glycosylated polypeptide chain of
5 585 amino acids of formula molecular weight 66,500. The amino acid sequence of HSA has been
established by protein sequence analysis (Meloun et al, 1975, "Complete amino acid sequence of human
serum albumin" FEBS. Letters: 58^1. 136-317; Behrens et al, 1975, "Structure of human serum albumin"
Fed. Proc. 34, 591) and more recently by genetic analysis (Lawn et al, 1981, Nucleic Acids Research 9,
6102-6114). Although there have been discrepancies between the amino acid sequences as published
10 (some being attributable to polymorphisms), Figure 1 represents the amino acid sequence currently
believed to be most representative of the HSA present within the human population.

Because of its relatively small molecular weight and net negative charge at physiological pH (Peters.
1970, "Serum albumin", Adv. Clin. Chem. 13, 37-111), HSA contributes 85% of the osmotic effect of normal
plasma. Thus HSA is the principal regulator of plasma volume. A secondary role of HSA is to bind small
is molecules produced by catobolic processes (for example fatty acids and bilirubin). Albumin represents the
principal means for the transport of these key metabolites, which are poorly soluble at physiological pH.
-nysical, chemical, immunological and limited proteolytic studies of HSA have shown that the molecule is
composed of regions of polypeptide chains which retain their conformation after separation from the parent
molecule by enzymatic means. These polypeptide chains retain their binding capabilities thereby facilitating
20 the mapping of binding sites for bilirubin, fatty acids and other small molecules to particular regions of the
polypeptide chain (Kragh-Hansen. 1981, "Molecular aspects of ligand binding to serum albumin". A. Soc.
Pharm. Expt. Ther. 33. 1, 17-53). Much of the information in this area has been reviewed (Brown and
Shockley. 1982, "Serum albumin: structure and characterisation of its ligand binding sites").

The indications for the clinical use of therapeutic concentrates of HSA are related principally to its
25 oncotic action as a plasma volume expander. Concentrates of HSA have been used therapeutically since
the 1940's. in particular in cases of shock, bums, adult respiratory distress syndrome, and cardiopulmonary
bypass. Albumin has also been used in cases of acute liver failure, following removal of ascitic fluid from
patients with cirrhosis, after surgery, in acute nephrosis, in renal dialysis, and as a transport protein for
removing toxic substances, such as in severe jaundice in haemolytic disease of the new born.
30 In addition to its use as a therapeutic agent HSA is a major component of serum added to media used
to support the growth of mammalian cells in tissue culture. The consumption of serum and hence of
albumin has been greatly increased over recent years as biotechnology and pharmaceutical companies
have expanded their tissue culture for research and for production. There is a universal need for lower cost
and better regulation of sera for these purposes.
35 It is known to manipulate the HSA-encoding DNA sequence express a recombinant polypeptide in
microorganisms. Indeed such a recombinant HSA polypeptide has been produced in bacterial species such
as Escherichia coli (G.B. Patent No. 2 147 903B) and Bacillus subtilis (European Patent Application No.
86304656.1) andThe yeast Saccharomyces cerevisiae (European Patent Publication No. 201 239. Delta
Biotechnology Ltd.); thus it is generally accepted that a recombinant polypeptide essentially identical to
40 natural HSA can be produced in a variety of microbial hosts by employing known methods. However, in all
cases where recombinant HSA has been produced, the objective has been to produce a molecule which is
"nature-identical" to HSA in structure and biological function.

It has now been found that it is advantageous to produce shorter forms of HSA.
One aspect of the present invention provides a polypeptide comprising the N-terminaJ portion of human
45 serum albumin up to amino acid residue n, where n is 369 to 41 9, and variants thereof.
The novel polypeptides of the invention are hereinafter referred to as "HSA(1-n)".
The terms "human serum albumin" is intended to include (but not necessarily to be restricted to)
known or yeMo-be discovered polymorphic forms of HSA. For example, albumin Naskapi has Lys-372 in
place of Glu-372 and pro-albumin Christchurch has an altered pro-sequence. The term "variants" is
50 intended to include (but not necessarily to be restricted to) minor artifical variations in residues 1 to n (such
as molecules lacking one or a few residues, having conservative substitutions or minor insertions of
residues, or having minor variations of amino acid structure). Thus polypeptides which have 80%, preferably
85%. 90%, 95% or 99%, homology with any HSA (1-n) compound are deemed to be "variants". Such
variants are preferably 360 to 430 amino acids long, more preferably 369 to 419 amino acids long and most
preferably 386 to 388 amino acids long. It is also preferred for such variants to be physiologically equivalent

EP 0 322 094 A1

to HSA (1-n) compounds; that is to say, variants preferably share at least one pharmacological utility with
HSA (1-n) compounds. Furthermore, any putative variant which is to be used pharmacologically should be
non-immunogenic in the animal (especially human) being treated.

Conservative substitutions are those where one or more amino acids are substituted others having

s similar properties such that one skilled in the art of polypeptide chemistry would expect at least the
secondary structure, and preferably the tertiary structure, of the polypeptide to be substantially unchanged.
For example, typical such substitutions include alanine or valine for glycine, arginine or asparagine for
glutamine, serine for asparagine and histidine for lysine. Variants may alternatively, or as well, lack up to ten
(preferably only one or two) amino acid residues in comparison with any given HSA (1-n); preferably any

to such omissions occur in the 100 to 369 portion of the molecule (relative to mature HSA itself). Similarly, up
to ten, but preferably only one or two, amino acids may be added, again in the 100 to 369 portion for
preference. The term "physiologically functional equivalents" also encompasses larger molecules compris-
ing the said 1 to n sequence plus a further sequence at the N-terminal (for example, pro-HSA(l-n). pre-pro-
HSA(1-n). met-HSA(1-n),and HSA(1-n) having a suitable leader sequence which is not necessarily native to

75 HSA).

If the HSA (1-n) is to be prepared by culturing a transformed yeast (S. cerevisiae ) as is described in
more detail below, the leader sequence may, for example, be that found naturally with the yeast alpha-
factor protein. C-terminal fusion products with other polypeptides of interest may be produced. Known
forms and fragments of HSA are clearly to be regarded as excluded from the above definition, for example

20 HSA(1-387), which was a peptic fragment produced in low yield (Geisow and Beaven, Biochem. J. 161. 619-
624. 1977 and ibid. 163. 477-484. 1977. These prior articles identify the fragment as 1-386, but it has since
become apparenT(see7 for example. Lawn et ai, op-cit .) that this is due to the authors 1 use of incorrect
published sequence information and that the fragment was in fact 1-387). Similarly, a C-terminal fusion
protein comprising HSA (1-n) and the remaining HSA residues (numbers n + 1 to 585) is not claimed as part

25 of the invention.

Particularly preferred novel HSA(1-n) compounds include HSA(1-373) (i.e. C-terminal Val). HSA(l-388)
(i.e. C-terminal He). HSA(1-389) (».e. C-terminal Lys). HSA(1-390) (i.e. C-terminal Gin) and HSA(1-407) (i.e.
C-terminal Leu).

The HSA(l-n) molecules are preferably produced by means for recombinant DNA technology
30 (optionally followed by proteolytic digestion), rather than by chemical or enzymatic degradation of natural
HSA, or by peptide synthesis. In the case of enzymatic degradation, for example, a trypsin-like enzyme will
cleave HSA between Lys(389) and Gln(390) but also concomitantly at other cleavage sites. In the future,
peptide synthesis may become more feasible for molecules as long as 419 amino acids, but at present is
not a practical proposition. Expression in yeast is particularly preferred.

35 It has been found that, at least in some situations where the HSA(1-n) compound is produced by
culturing a transformed host some HSA(1-n) compounds which are longer than HSA(1-387) are prot-
eolytically digested back to HSA (1-387) by the enzymes which are naturally present in the system. Thus,
one can, if desired, use a nucleotide sequence corresponding to a given HSA(1-n) compound in order to
prepare another HSA(1-n) compound.

40 The new molecules herein described can be used as an effective substitute for either natural HSA or
nature-identical recombinant HSA as a plasma volume expander. An advantage of HSA(1-n) over natural
HSA and recombinant nature-identical HSA relates to the efficacy of raising the colloid osmotic pressure of
blood. The smaller molecular weight (approximately 44 kilo-daltons) of the protein of the present invention
means that an individual protein dose of only one-half to two-thirds that of natural-HSA or nature-identical

45 recombinant HSA will be required for the equivalent colloid osmotic effect. Consequently, any process for
the production of this novel polypeptide by means of recombinant DNA technology may afford significant
economic advantages over known processes for the production of nature-identical recombinant HSA. since
substantially less proteinaceous material is required to be produced for an effective dose.

Thus, a second aspect of the invention provides a pharmaceutical composition comprising HSA(1-n)-

50 plus, where HSA(1-n)plus is HSA(1-n) as defined above or any HSA(1-n) molecules which are known per se
but have not been proposed for pharmaceutical use:

HSA (1-387) which, as discussed above, was a fragment produced by chance in a prior art peptic
digest of HSA, is particularly preferred as the HSA(1-n) plus in such a pharmaceutical composition. The
composition may comprise "variants" of HSA (1-387) as defined above.

55 A third aspect provides a method of treating a human for shock, burns or other conditions in which
albumin is indicated, comprising administering intravenously a blood-bulking or blood-clearing effective non-
toxic amount of a sterile non-pyrogenic solution of a polypeptide comprising HSA(l-n) plus.

Further aspects of the invention include (a) vectors, plasmids and transformed microorganisms,

EP 0 322 094 A1

including cell lines, encoding HSA(l-n)plus expression; (b) processes for the production of HSA(1-n)plus
comprising the fermentation under suitable conditions of a microorganism (including a cell line) so
transformed as to express HSA(1-n)plus; and (c) laboratory media comprising HSA(l-n)plus.

A futher advantage of at least some HSA(1-n) plus molecules over nature-identical recombinant HSA is

5 that their smaller size and thus reduced amino acid content has been found to lead to an increase in the
yield obtained (molecules per cell dry weight) in microbial hosts relative to that obtained currently for
nature-identical recombinant HSA. Thus, not only has it been found that the scale of the process can be
reduced, but also productivity in the recombinant host organism can be enhanced.

The compounds of the invention may be used as- blood-bulking (plasma-expanding) agents in analo-

w gous ways and in analogous formulations as HSA itself except that the dose of the HSA(1-n)plus compound
(in terms of weight) will generally be less than that of HSA as the oncotic effect of the former is greater. The
pharmacist or clinician skilled in the art will readily be able to determine by routine and non-inventive
experimentation the optimum dose of the HSA(1-n)plus compound. Generally, the amount of HSA(1-n)plus
which is administered will be about two-thirds of the amount of HSA which would be administered.

75 HSA (1-n) plus compounds may also be used as:

(1) substitutes for HSA or, more commonly, bovine serum albumin (BSA) in tissue culture media, thereby
reducing the risk of contamination of the medium with, for example, viruses and mycoplasmas; (2)
substitutes for BSA in the stationary phase in liquid chromatography for resolution of enantiomers and so
on.

20 '

EXAMPLES

25 The invention will now be Illustrated by way of example and with reference to the drawings, in which:

Figure 1 depicts the amino acid sequence currently .thought to be the most representative of natural
HSA. with (boxed) the alternative C-terminal of HSA(1-n);

Figure 2 depicts the DNA sequence coding for mature HSA;
Figure 3 illustrates, diagrammatically. the construction of mHOB16;
30 Figure 4 illustrates, diagrammatically, the construction of pHOB31; and

Figure 5 is a copy of a rocket electrophoretogram showing the increased yield of HSA(1-389) over
complete HSA.

Standard recombinant DNA procedures are as described by Maniatis et al (1982) unless otherwise
35 stated. Construction and analysis of M13 recombinant clones was as described by Messing (1983) and
Sanger et al. (1977).

The~human serum albumin coding sequence used in the construction of the following molecules is
derived from the plasmid M13mp19.7 (European Patent Application No. 201 239. Delta Biotechnology Ltd.)
or by synthesis of oligonucleotides equivalent to parts of this sequence. Oligonucleotides were synthesised
40 using phosphoramidite chemistry on an Applied Biosystems 380B oligonucleotide synthesizer according to
the manufacturer's recommendations (AB Inc.. Warrington. Cheshire, England).

Example 1: HSA (1-389)

An expression vector was constructed in which DNA encoding the HSA secretion signal and mature
HSA up to and including the 389th amino acid, lysine, was placed downstream of the S.cerevisiae
phosphoglycerate kinase gene ( PGK) promotor and followed by a stop codon and the PGK terminator of
transcription. This vector was then introduced into S.cerevisiae by transformation and directed the expres-
50 sion and secretion from the cells of a molecule representating the N-terminal 389 amino acids of HSA.

An oligonucleotide was synthesised (Linker 1) which represented a part of the known HSA coding
sequence (Figure 2) from the Pstl site (1092, Figure 2) to the codon for valine 381 wherein that codon was
changed from GTG to GTC:

Linker 1

EP 0 322 094 A1

DPHECYAKVF D E
5' GAT CCT CAT GAA TGC TAT GCC AAA GTG TTC GAT GAA

3' ACGT CTA GGA GTA CTT ACG ATA CGG TTT CAC AAG CTA CTT
5 1100 1120

F K P L V
to TTT AAA CCT CTT GTC 3 1

AAA TTT GGA GAA CAG 5'

Linker 1 was ligated into the vector M13mp19 (Norrander et al, 1983) which had been digested with Pstl
is and Hindi and the ligation mixture was used to transfect l.coli strain XLVBlue (Stratagene Cloning
Systems. San Diego. CA). Recombinant clones were identified by their failure to evolve a blue colour on
medium containing the chromogenic indicator X-gal (5-bromo^chloro-3-indolyl-0-D-galactoside) in the
presence of IPTG (isopropylthto-0-gaIactoside). DNA sequence analysis of template DNA prepared from
bacteriophage particles of recombinant clones identified a molecule with the required DNA sequence,
20 designated mHOB1 2 (Figure 3).

M13mp19.7 consists of the coding region of mature HSA in M13mp19 (Norrander et al. 1983) such that
the codon for the first amino acid of HSA. GAT. overlaps a unique Xhol site thus:

Asp Ala

25 5' ' C'*T C G A G 'a T G C A 3 1

3' G A G C T A C .T ACGT 5'
Xho l

(EPA No. 210239 A1). M13mp19.7 was digested with Xho l. made flush-ended by S1 -nuclease treatment
and was then ligated with the following oligonucleotide (Linker 2):

35 Linker 2

5 , TCTTTTATCC

'a+A G C T t'g GATAAAAGA3'

3' AGAAAATAGGTTCG A iA C CTATTTTCT5*

I— -1— I

Hind lll

46 The ligation mix was then used to transfect E.coli XL1-Blue and template DNA was prepared from
severai plaques and then analysed by DNA sequencing to identify a clone. pDBD1 (Figure 4). with the
correct sequence.

A 1.1 kb Hindlll to Pst1 fragment representing the 5 end of the HSA coding region and one half of the
inserted oligonucleotide linker was isolated from pDBD1 by agarose gel electrophoresis. This fragment was

50 then ligated with double stranded mHOB12 previously digested with Hind lll and Pstl and the ligation mix
was then used to transfect E.coli XL1-Blue. Single stranded template DNA was prepared from mature
bacteriophage particles of several plaques. The DNA was made double stranded in vitro by extension from
annealed sequencing primer with the Klenow fragment of DNA polymerase I in the presence of deox-
ynucleoside triphosphates. Restriction enzyme analysis of this DNA permitted the identification of a clone

55 with the correct configuration. mHOB15 (Figure 4).

The following oligonucleotide (Linker 3) represents from the codon for the 382nd amino acids of mature
HSA (glutamate, GAA) to the codon for lysine 389 which is followed by a stop codon (TAA) and a Hindlll
site and then a BamHI cohesive end:

EP 0 322 094 A1

Linker 3

EEPQNLIKJ
5* GAA GAG CCT CAG AAT TTA ATC AAA TAA GCTTG 3 1
3' CTT CTC GGA GTC TTA AAT TAG TTT ATT CGAACCTAG 5'

This was ligated into double stranded mHOBiS, previously digested with Hindi and Bam HI. After
ligation, the DNA was digested with Hind i to destroy all non-recombinant molecules and then used to
transfect E.coli XL1-Blue. Single stranded DNA was prepared from bacteriophage particles of a number of
clones and subjected to DNA sequence analysis. One clone having the correct DNA sequence was
designated mHOBl6 (Figure 4).

A molecule in which the mature HSA coding region was fused to the HSA secretion signal was created
by insertion of Linker 4:

Linker 4

M , K W V S F I 5 L L F L

5' GATCC ATG AAG TGG GTA AGC TTT ATT TCC CTT CTT TTT CTC

G TAC TCC ACC CAT TCG AAA TAA AGG GAA GAA AAA GAG

FSSAYSRGVFRR
TTT AGC TCG GCT TAT TCC AGG GGT GTG TTT CG 3 1

AAA ACG AGC CGA ATA AGG TCC CCA CAC AAA GCAGCT 5'

into BAM HI and Xhol digested Ml3mp19.7 to form pDBD2 (Figure 5). In this linker the codOn for the fourth
amino acid after the initial methionine, ACC for threonine in the HSA pre-pro leader sequence (Lawn et al.
1981). has been changed to AGC for serine to create a Hindlll site.

The 5* end of this construction was removed as a Bam HI to Pvull fragment and ligated with the Pvull to
Bam HI fragment of double stranded mHOB16 (representing the 3 end of the truncated HSA gene) into
pMA91 (Mellor et al, 1983) at the Bglll site to form pHOB31 (Figure 4). This molecule contains the truncated
HSA coding region with the HSA secretion signal between the S.cerevisiae PGK gene promoter and
terminator such that the 5' end of the gene abuts the promoter. The molecule also contains a selectable
marker for yeast transformation. LEU2 , and part of the yeast 2um plasmid to permit autonomous replication
in yeast

The plasmid pHOB31 was introduced into S.cerevisiae AH22 (Hinnen et al, 1978) by transformation
using standard procedures (Beggs, 1978). Purified transformants were grown in YEPD broth (1% yeast
extract. 2% peptone, 2% glucose) for 3 days at 30 *C and the culture supernatant was then analysed,
successfully, for the presence of HSA-related material by rocket gel electrophoresis. Figure 5 shows the
electrophoretogram;: the yield of HSA-related material from transformants harbouring a plasmid encoding
HSA(1-389) is demonstrably higher than the yield from a transformant secreting mature, natural, HSA

However, production of HSA (1-389) gave a product indistinguishable from HSA (1-387) (see Example
2) by both amino-terminal and carboxy-terminal sequence analysis. This is probably explained by the
efficient removal of the COOH-terminal sequence lle-Lys.

EXAMPLE 2: HSA (1-387)

The construction of a plasmid encoding HSA (1-387) was identical to the procedure for construction of
the HSA (1-389) plasmid, pHOB31, except that the linker 3 was substituted by linker 5 (shown below) which
represents the region from the codon for the 382nd amino acid of mature HSA (glutamate, GAA) to the

EP 0 322 094 A1

codon for leucine 387 which is followed by a stop codon and a Hindlll site and then a Bam HI cohesive end:

Linker 5

Stop

GAA

GAG

CCT

CAG

AAT

TTA

TAA GCTTG 3'

CTT

CTC

GGA

GTC

TTA

AAT

ATT CGAACCTAG 5'

The remainder of the construction was as detailed above for pHOB31 and resulted in the plasmid pDBDS.
, 5 EXAMPLE 3: (1-369)

In order to construct a plasmid encoding HSA (1-369), a linker was synthesised representing the region
from the Pstl site of mature HSA (position 1092, Figure 3) to the codon for cystine 369 which was followed

Linker 6

Stop

GAT

CCT

CAT

GAA

TGC

TAA GCTTG

A CGT CTA

GGA

GTA

CTT

ACG

ATT CGAACCTAG

This linker was ligated with the BamHI Pstl fragment of pDBD2. representing the 5 part of preproHSA,
into pMA91 at the Bglll site. Aplasmid with the correct configuration was termed pDBD3 (Figure 6).

Production of HSA (1-369) by culturing S.cerevisiae transformed with pDBD3 gave low yields, indicating
that the product may have been unstable in the yeast expression system used.

EXAMPLE 4: HSA (1-419)

For the construction of a plasmid encoding HSA (1-419) the Bam HI - Hindi fragment of pDBD2 was
ligated with an annealed self-complementary oligonucleotide (linker 7):

Unker 7

5' ATAAGCTTGGATCCAAGCTTAT 3'

and then the ligation mix was digested with Bam HI and the fragment was ligated into pMA91 to give pDBD4
(Figure 7). In this construct the Hindi site (1256, Figure 3) of pDBD2 creates a blunt end after the second
base of the codon for serine 419 and this codon is reformed by the linker 6 such that this codon is followed
by a stop codon, a Hindlll site and a Bam HI site.

Expression of HSA (1-419) via plasmid pDBDS in S.cerevisiae produced a molecule with the correct

amino terminal sequence (Asp-Ala-His ) but leucine and not serine was the COOH-terminal residue.

Attempts to isolate the COOH-terminal peptide using a covalent label which should attach to cysteine 392
also were unsuccessful. It was concluded that proteolysis of part of the COOH-terminus of HSA (1-419)
occurred. This is consistent with the observation of a small percentage of proteolysis in the same position of
full-length HSA produced in an analogous manner in yeast (Sleep et al. 1988).

EXAMPLE 5: Fermentation of HSA(1 -n)plus-producing yeast

EP 0 322 094 A1

A laboratory fermenter is filled to half its nominal working volume with an initial "batch" medium
containing 50ml/l of a salts mixture (containing 114g/l KH 2 POi, 12g/l MgSO* . 3.0g/l CaCI 2 .6H 2 0, 2.0g/l Na 2
EDTA: lOml/l of a trace elements solution containing 3g/l ZnS04.7H 2 0. 10g/l FeSO*.7H 2 0, 3.2g/l
MnS0 4 .4H 2 0. 79mg/l CuSO*.5H 2 0, 1.5g/l H 3 B0 3 , 0.2g/l Kl, 0.5g/l Na 2 MoO*.2H 2 O.0.56g/l CoCI 2 .6H 2 0,
5 75ml/l H3PO4: 20g/l sucrose: 50ml/l of a vitamins mixture containing 1.6g/l Ca pantothenate, 1.2g/l nicotinic
acid, 12.8g/l m inositol, 0.32g/l thiamine HCI and 8mg/l pyridoxine HCI and 8mg/l biotin. An equal volume of
"feed" medium containing 100ml/l of the salts mixture, 20ml/l of trace elements solution 500g/l sucrose and
100ml/l vitamin solution is held in a separate reservoir connected to the fermenter by a metering pump.

10 The fermenter is inoculated with Saccharomyces cereyislae which has been transformed as above with
plasmid pDBD3 from Example 2. The pH is maintained at 5.7 t 0.2 by automatic addition of ammonia or
sulphuric acid, the temperature is kept at 30 *C and the stirred speed is adjusted to give a dissolved
oxygen tension (DOT) of > 20% air saturation at 1 v/v/min air flow rate. When the initial substrate has been
consumed, the metering pump is turned on, maintaining a growth rate of approximtely 0.1 Sh" 1 . The pump

75 rate is increased to maintain this growth rate until the stirrer speed reached its maximum value at which
point it is not possible to increase the pump rate any further without causing the DOT to fall below 15% air
saturation which is the minimum value permitted to occur. PPG 2000 is added in response to a foam
sensor. None is added until over 50% of the feed solution had been added. The final level of. addition is
0^g/l.

20 HS A(1 -387) is secreted into the medium

EXAMPLE 6: Binding of bilirubin to HSA(1-387)

25 Binding of the haem metabolite, bilirubin, to HSA (1-387) was carried out by a fluorescence enhance-
ment method (Beaven and Gratzen (1973) Eur. J. Biochem. 33, 500-510). Figure 8 shows that the
enhancement of bilirubin fluorescence as a function of protein/bilirubin ratio is indistinguishable for HSA(1-
387) and clinical grade HSA.

The interaction of HSA and bilirubin is very sensitive to the conformation of the protein (Beaven and

30 Gratzen, loc. cit.) and these results indicate that no gross alteration in conformation of the regions of HSA
represented~by"HSA(1-387) has occurred through the expression of a shorter molecule.

EXAMPLE 7: Oncotic behaviour of HSA(1-387)
35 ~~

HSAO-387) was concentrated in 0.9% w/v saline to a final protein concentration of 54 mgmU Dilutions
of this concentrate, together with dilutions of a clinical grade HSA (100 mg/ml), were compared for osmotic
effect in a colloid osmometer. Figure 9 indicates that HSA(1-387) gives a colloid osmotic pressure
approximately one-third higher than that of full-length HSA at a given protein concentration. Importantly, the
AO increase in colloid osmotic pressure with protein concentration is approximately linear over a range up to
5% w/v, which represents the concentration in plasma.

This indicates that HSA(1-387) does not self-associate appreciably within a useful working clinical
concentration range.

EXAMPLE 8: Formulations for injection

The HSA(1-n)plus of the invention may be presented in container sizes ranging from 20ml to 500ml,
with the concentration thereof varying (typically) from 2% to 17%, for example 3%, 13% or 17%.
so The solution for administration is sterile and pyrogen free. A 3% solution is osmotically similar to human
plasma. At least 96% of the total protein is preferably albumin. The sodium ion content is generally between
130-1 60mmol/litre and the potassium ion content is generally not more than 2mmol/litre. The pH is adjusted
to 6.9 * 0.5. The concentration of citrate is generally no more than 20mmol/iitre and may be absent
altogther.

55 Stabilizers may be used, for example either 0.16 millimole sodium acetyl tryptophanate, or 0.08
millimole sodium acetyl tryptophanate and 0.08 millimole sodium caprylate per gram of HSA(1-n)plus.

EP 0 322 094 A1

References

i i —

Beggs, J.D. (1978). Nature. 275, 104-109.

Brown. J.R. and Shockley, P.. (1982) in "Upid-Protein Interactions" 1, 25-68. Eds. Hayes, 0. and Jost, P.C.
5 Hinnen, A. et al (1978). Proc. Natl. Acad. Sci. USA, 75, 1929-1933. "
Lawn, R.M. et al (1981). Nucl. Acid. Res. 9, 6103-6114.

Maniatis. T. et al (1982). Molecular cloning: A laboratory manual. Cold Spring Harbour Laboratory, Cold
Spring Harbor, New York.
Meilor, J. et al (1983). Gene, 24, 1-14.
w Messing, J. (1983). Methods Enzymol. 101, 20-78.
Norrander, J. et al (1983). Gene, 26, 101-106.
Sanger, F. et al (1977). Proc. Natl. Acad. Sci. USA, 74, 5463-5467.
Sleep. D. Belfield. G.P. and Goodey, A.R. (1988) Yeast 4, S168.

Claims

I. A polypeptide comprising the N-terminal portion of mature human serum albumin up to amino acid
residue n. where n is 369 to 419. but not 387, and variants thereof.

20 2. A polypeptide according to Claim 1 wherein the polypeptide is selected from the group consisting of
HSA (1-373), HSA (1-388), HSA (1-389), HSA (1-390) and HSA (1-407) and variants thereof.

3. A pharmaceutical composition comprising a polypeptide according to Claim 1 or 2 except that n may
be 387.

4. A composition according to Claim 3 wherein the polypeptide is HSA (1-387) or a variant thereof.

25 5. A nucleotide sequence encoding a polypeptide comprising the N-terminal portion of mature human
serum albumin up to amino acid residue n, where n is 309 to 419, and polypeptide variants thereof, the
nucleotide sequence not being linked at its 3' end to a further sequence encoding the C-terminal portion of
mature human serum albumin from amino acid residue n + 1 to 585.
6. A nucleotide sequence according to Claim 5 wherein n is 387.

30 7. A nucleotide sequence according to Claim 5 or 6 linked at its 5 end to a further nucleotide sequence
encoding a peptide corresponding to the pro-, pre-, or pre-pro- position of HSA, a methionine residue, or
another leader sequence.

8. An expression vector suitable for transformation of and expression in a selected host the vector
comprising a nucleotide sequence according to any one of Claims 6 and 8 and the said nucleotide

35 sequence being a DNA sequence.

9. A host organism transformed with a vector according to Claim 8.

10. A host organism according to Claim 9 which is Saccharomyces cerevisiae .

II. A process for the production of a polypeptide comprising the culture under suitable conditions of a
host microorganism according to Claim 9 or 10, the said polypeptide being encoded by the said nucleotide

40 sequence.

12. A laboratory medium for the growth of microorganisms comprising a polypeptide according to Claim
1, except that n may be 387.

13. A medium according to Claim 12 wherein n is 387.

EP 0 322 094 A1

FIGURE 1

I - N3L 1 -firioerelch* / Mowly filed
Nouvellement dSpoB§ ■

10 20
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys

30 40
Ala Leu Val Leu lie Ala Phe Ala Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val

50 60
Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu

70 80
Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

90 100
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu

110 120
Cys Phe Leu Gin His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 140
Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr

150 160
Glu He Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

170 180
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro

190 200
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys

210 220
Ala Ser Leu Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser

230 240
Gin Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

250 260
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu

270 280
Ala Lys Tyr He Cys Glu Asn Gin Asp Ser He Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 300
Lys Pro Leu Leu Glu Lys Ser His Cys lie Ala Glu Val Glu Asn Asp Glu Met Pro Ala

310 320
Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

330 340
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp

350 360
Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 380
Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu

EP 0 322 094 A1

"52 K**

FIGURE 1 Cont,

390 400
Val Glu Glu Pro Gin Asn Leu lie Lys Gin Asn Cys Glu Leu Phe Glu Gin Leu Gly Glu

410 420
Tyr Lys Phe Gin Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr

430 440
Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 460
Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gin Leu

470 480

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

490 500
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys

510 520
Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp He Cys Thr Leu Ser Glu Lys Glu

530 540
Arg Gin He Lys Lys Gin Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

550 560
Lys Glu Gin Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

570 580
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gin

Ala Ala Leu Gly Leu

EP 0 322 094 A1

FIGURE 2 DNA sequence coding for mature HSA

10 20 30 40 50 60 70 80

GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTT
DAHKSEVAH RFKD L G E E N F KAL VL IAF

90 100 110 . 120 130 140 150 160

TGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATC^TGTAAAATTAGTGAA

AQYLQQCPFED HVKL VNEVTEFARTC

170 180 190 200 210 220 230 240

TTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTT
VADESAENC DKS LHTLFGDKLCTVATL

250 260 270 280 290 300 310 320

CGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGA
RETYGEMADCCAKQEPERNECFLQHKD

330 340 350 360 370 380 390 400

TGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTT^^

DNPNLPRLVRPEVDVMCTAFHDNEET

410 420 430 440 450 460 470 480

TTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTC
FLKKYLYE IARRHPYFYAPELLFFAKR

490 500 510 520 530 540 550 560

TATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGA
YKAAFTECC Q A A D KAACL L P K L D E LRD

570 580 590 600 610 620 630 640

TGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAATGTGCCAGTCTCCAAAAATO

EGKASSAKQRLKCASLQKFGERAFKA

650 660 670 680 690 700 710 720

GGGCAGTGGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAA
WAVARLSQ RFPKAEFAEVS K L V T D LTK

730 740 750 760 770 780 790 800

GTCCAraCGGAATGCTGCCATGGAGATCTGCTTGAATGT
VJ^TECCHGD LLECADDRADLAKY I C E N

810 820 830 840 850 860 870 880

TCAGGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGG
QDS I S SRLKE CCEKPL LEKS H C I A E V

890 900 910 920 930 940 950 960

AAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA

ENDEMPADLPSLAADFVES KDVCKNYA

970 980 990 1000 1010 1020 1030 1040

GAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCT
EAKDVF LGMFLYEYARRHPDYSVVLLL

| IVuj oinaereiohl / Nsw;/ 'led

tieu eLnqerelehii-Mcw^y fil'Scj
ep o 322 094 A1 1 Noir'eali *nnnt #p' <s£

FIGURE 2 Cont ,

1050 1060 1070 1080 1090 1100 1110 1120

GAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGT
RLAKTYETTLEKCCAAADPHECYAKV

1130 1140 1150 1160 1170 1180 1190 1200

TCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAACTGTGAGCTTTTTGAGCAGCTTGGAGAG
FDEFKPLVEEPQNLIKQN CELFEQLGE

1210 1220 1230 1240 1250 1260 1270 1280

TACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTC
YKFQNALLVR YTKKVPQVSTPTLVEVS

1290 1300 1310 1320 1330 1340 1350 1360

AAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTAT
RNLGKVGSKCCKHPEAKRMPCAED YL

1370- 1380 , 1390 1400 1410 1420 1430 1440

CCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACAAAATGCTGCACAGAGTCC
SVVLNQLCVLHEK TPVSDRVTKCCTES

1450 1460 1470 1480 1490 1500 1510 1520

TTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATT
L'VNRRP C FSALEVDET YVPKEFN AETF

1530 1540 1550 1560 1570 1580 1590 1600

CACCTTCCATGCAGATATATGCACACTTTCT^

TFHAD ICTLSEKERQIKKQTALVEL V

1610 1620 1630 1640 1650 1660 1670 1680

AACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGA

KHKPKATKEQLKAVMDDFAAFVEKCCK

1690 1700 1710 1720 1730 1740 1750 1760

GCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATAACA
A D D K E TCFAEEGKKLV AA5QAALGL

1770 1780
TCTACATTTAAAAGCATCTCAG

ep 0 322 094 ai i Kcj t irr,is'eic'»t i Ns*rty 'wed ,
! Nojvolleme.-.t d^ose t

FIGURE 3 Construction of mH0B16

EP 0 322 094 A1

FIGURE 4 Construction of pH0B3]

FIGURE 5

I 2 3 +. S 6 7 ?

Rocket immunoelectrophoretic analysis of culture supernatant from
S .cerevisiae AH22 trans formants obtained with a plasmid containing the
complete HSA coding region (samples 1-4) and from transf ormants harbouring
an equivalent plasmid encoding truncated HSA (1-389) (samples 5-8).

EP 0 322 094 A1 J O i.,ijo r0 | ;.V I f> ev.'ly U\ ■

FIGURE 6 Construction of pDBD3

FIGURE 7 Construction of pDBD4

EP 0 322 094 A1

Digest with
BamHI and Hindi

pDBD2
BamHI- Hind i
fragment

B = BamH I; Bg = Bglll;
H = Hindl ll; He » Hind i;
P = PstI

H B H

Linker 7
(blunt ended)

Ligate then digest
with BamHI

Ligate

pMA9 1 digested with
Belli

EP 0 322 094 A1

FIGURE 8

EP 0 322 094 A1

Meu eingerei
NbuveHemei

ohtf Newly f iled

-,t d6po

aV5

FIGURE 9

5 10 15 20 25 30 35 40 45 50

PROTEIN (mg/ml)

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Office

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Application Nomber

EP 88 31 0000

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